Methods for inhibiting cutaneous inflammation and hyperpigmentation

ABSTRACT

This invention provides a method of preventing or treating in a subject contact dermatitis which comprises administering to the subject an amount of a compound capable of inhibiting the stem cell factor signaling pathway effective to prevent or treat contact dermatitis so as to thereby prevent or treat contact dermatitis in the subject. This invention also provides a method of preventing or treating in a subject hyperpigmentation, asthma, cutaneous inflammation, anaphylaxis and bronchospasm, mastocytosis, tumors which express activated kit, and conception.

This application is a §371 national stage of PCT InternationalApplication No. PCT/US00/12405, filed May 5, 2000, designating theUnited States of America, which is a continuation-in-part and claimspriority of U.S. Ser. No. 09/474,478, filed Dec. 29, 1999, which is acontinuation-in-part of U.S. Ser. No. 09/306,143, filed May 6, 1999 nowU.S. Pat. No. 6,576,812, the contents of which are hereby incorporatedby reference.

Throughout this application, various publications are referenced byarabic numerals within parentheses. Disclosures of these publications intheir entireties are hereby incorporated by reference into thisapplication to more fully describe the state of the art to which thisinvention pertains. Full bibliographic citations for these referencesmay be found immediately preceding the claims.

The invention described herein was made with Government support undergrant numbers 1 R29 AR 40514-01A1, 5 P30 041942 and 1-RO1-AR43356-01A2from the National Institutes of Health. Accordingly, the United StatesGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

The use of murine models to investigate human cutaneous oncology,immunology and keratinocyte biology is advantageous over the use ofhuman skin for obvious reasons. However, substantial differences existbetween human skin and murine skin. In human skin, Stem Cell Factor isproduced by epidermal keratinocytes after birth, unlike in normal murineskin. The result of this, among other things, is that melanocytes arepresent in the interadnexal epidermis in human skin. In contrast,melanocytes in adult murine skin are generally confined to hairfollicles, with the exception of rare epidermal melanocytes found in theears, footpads, and tail (1). A few dermal melanocytes may also be foundin mice, mostly in the ears. These differences have compromised the useof the mice as a model system for investigation of human cutaneousbiology.

It has been discovered that melanocyte migration and development, aswell as the survival of melanocytes and mast cells, are dependent onexpression of the kit protein, a receptor tyrosine kinase encoded by thec-kit proto-oncogene (2–6). The ligand for kit, known as stem cellfactor (SCF) (also called mast cell growth factor, steel factor, and kitligand) may be produced locally in human skin by epidermalkeratinocytes, fibroblasts, and endothelial cells (7–8). However,definitive studies of SCF production in murine skin have not beenreported. Transgenic studies using the SCF gene promoter region andbeta-galactosidase as a reporter gene suggest that, unlike in humanskin, postnatal murine cutaneous SCF expression is limited to the dermisand hair follicles, and not found in epidermal keratinocytes (9). Thedifference in SCF expression between human and murine epidermis couldexplain the difference in melanocyte distribution and other biologicalphenomena in these two species.

SCF may be produced in two isoforms by alternate splicing of exon 6. Oneisoform lacks exon 6 encoded sequences and exists predominantly as amembrane-bound molecule. The other isoform contains exon 6 encodedsequences which include a protease sensitive site (10–19). Cleavage atthe protease sensitive site causes the release of a soluble, bioactiveform of SCF. The membrane-bound and soluble forms of SCF havedifferential effects on melanocyte precursor dispersal and survival (20)and exogenous soluble SCF may produce—cutaneous mast cell hyperplasiaand cutaneous hyperpigmentation (21–23). In addition, local highconcentrations of soluble SCF have been found in lesions of humancutaneous mastocytosis, a disease characterized by dermal accumulationsof mast cells and increased epidermal melanin (7, 8, 24) and inspongiotic dermatitis, a common inflammatory condition of human skin(our unpublished data).

SUMMARY OF THE INVENTION

This invention provides a method of preventing or treating in a subjectcontact dermatitis which comprises administering to the subject anamount of a compound capable of inhibiting the stem cell factorsignaling pathway effective to prevent or treat contact dermatitis so asto thereby prevent or treat contact dermatitis in the subject.

This invention provides a method of preventing or treating in a subjecthyperpigmentation which comprises administering to the subject an amountof a compound capable of inhibiting the stem cell factor signalingpathway effective to prevent or treat hyperpigmentation so as to therebyprevent or treat hyperpigmentation in the subject.

This invention provides a method of preventing or treating in a subjectasthma which comprises administering to the subject an amount of acompound capable of inhibiting the stem cell factor signaling pathwayeffective to prevent or treat asthma so as to thereby prevent or treatasthma in the subject.

This invention provides a method of preventing or treating in a subjectcutaneous inflammation which comprises administering to the subject anamount of a compound capable of inhibiting the stem cell factorsignaling pathway effective to prevent or treat cutaneous inflammationso as to thereby prevent or treat cutaneous inflammation in the subject.

This invention provides a method of preventing or treating in a subjectanaphylaxis and bronchospasm which comprises administering to thesubject an amount of a compound capable of inhibiting the stem cellfactor signaling pathway effective to prevent or treat anaphylaxis andbronchospasm so as to thereby prevent or treat anaphylaxis andbronchospasm in the subject.

This invention provides a method of preventing or treating in a subjectmastocytosis which comprises administering to the subject an amount of acompound capable of inhibiting the stem cell factor signaling pathwayeffective to prevent or treat mastocytosis so as to thereby prevent ortreat mastocytosis in the subject.

This invention provides a method of preventing or treating in a subjecturticaria which comprises administering to the subject an amount of acompound capable of inhibiting the stem cell factor signaling pathwayeffective to prevent or treat urticaria so as to thereby prevent ortreat urticaria in the subject.

This invention provides a method of preventing or treating in a subjecthypersensitivity reactions which comprises administering to the subjectan amount of a compound capable of inhibiting the stem cell factorsignaling pathway effective to prevent or treat hypersensitivityreactions so as to thereby prevent or treat hypersensitivity reactionsin the subject.

This invention provides a method of preventing or treating in a subjectairway inflammation which comprises administering to the subject anamount of a compound capable of inhibiting the stem cell factorsignaling pathway effective to prevent or treat airway inflammation soas to thereby prevent or treat airway inflammation in the subject.

This invention provides a method of preventing or treating in a subjectinterstitial cystitis which comprises administering to the subject anamount of a compound capable of inhibiting the stem cell factorsignaling pathway effective to prevent or treat interstitial cystitis soas to thereby prevent or treat interstitial cystitis in the subject.

This invention provides a method of preventing or treating in a subjecta tumor which expresses activated kit which comprises administering tothe subject an amount of a compound capable of inhibiting the stem cellfactor signaling pathway effective to prevent or treat a tumor whichexpresses activated kit so as to thereby prevent or treat a tumor whichexpresses activated kit in the subject.

The invention provides a method of providing contraception to a subjectwhich comprises administering to the subject an amount of a compoundcapable of inhibiting the stem cell factor signaling pathway effectiveto prevent conception so as to thereby provide contraception to thesubject.

This invention provides a method of desensitizing a subject to an agentwhich comprises administering to the subject an amount of a compoundcapable of inhibiting the stem cell factor signaling pathway effectiveto desensitize the subject to the agent so as to thereby desensitize thesubject to the agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1:

Transgene design. Both transgenes used the human keratin 14 promoter andpolyadenylation sequences. Transgene one included a rabbit b-globinintron, and transgene two included human growth hormone sequences toprovide for stability. Neither the beta globin intron nor the humangrowth hormone sequences produce protein products.

FIG. 2:

Increased mast cells in mice expressing epidermal membrane and solubleSCF (transgene one). (a) Numerous mast cells are demonstrated in thesuperficial dermis of body wall skin of newborn mice bearing transgeneone (membrane/soluble SCF), using an immunoperoxidase/alcian bluetechnique which stain mast cell granules metachromatically purple. Notethe apposition of mast cells (arrowheads) to basilar keratinocytes, thesource of SCF. Immunoperoxidase with an anti-S100 antibody in thispreparation also demonstrates melanocytes as brown staining cells in theepidermal basilar layers of epidermis and follicles (white arrows).Sebocytes are seen as large, round, lightly S-100(+) cells in thefollicular epithelium. Melanin pigment is stained black in thispreparation. (b) Immunofluorescence with anti-kit antibodies highlightskit expressing dermal mast cells (arrowheads) in body wall skin ofnewborn (transgene one membrane/soluble SCF) mouse. (c) Anti-kitantibody immunofluorescence shows mast cells crowded in the papillarydermis and extending into the upper reticular dermis and body wall skinof 21 day old, transgene one positive mouse, MC, confluent mast cells;arrowheads, individual and small clusters of mast cells; E, epidermis;F, follicles; K, keratin layer. (d) Hematoxylin and eosin-stainedsections show mast cells filling the superficial corium in section oftongue from a 21 day old, transgene one positive mouse. The lack ofabundant melanocytes and melanophages in this anatomic site allows easyvisualization of the mast cells. This histologic picture is identical tothat seen in human cutaneous mastocytosis. (e) Alcian blue stainedserial section of tongue shows metachromatic granules in mast cells of21 day old, transgene one positive mouse.

FIG. 3:

Electron microscopy confirms the presence of melanocytes and mast cellsin transgenic mice. (a) Transgene one mouse with membrane/solubleepidermal SCF has numerous dermal mast cells (arrowheads) as well asdermal melanocytes (arrows). Asterisks show the boundary of the dermisand hair follicle. Higher magnification images of mast cell andmelanocyte are shown in b and c, respectively. Original magnifications:(a) 2,750, (b) 9,000, (c) 11,750.

FIG. 4:

Transgenic phenotypes are stable across a wide range of gene expressionlevels. This figure compares the transgene copy number determined byPCR, with SCF mRNA expression as determined by RNase protection assay,in lines from different founders. The relative density of SCF bands wasdetermined by dividing the mean density of the SCF band by the densityof a SCF band derived from an identical aliquot of RNA. Probe templateswere 384 bases in length for SCF (40 base pairs of promoter sequence and342 bases complimentary to nucleotides 814–1156 of murine SCF mRNA (5).A beta-actin probe was used as a control, and to allow standardizationbetween RNA preparations from different mice. The beta-actin probelength was 310 bases, 227 bases of which are complementary to murinebeta-actin mRNA. The probe was purchased from Ambion(pTR1-beta-actin-mouse anti-sense control template). Note thedifferences between TG2 (4×, 5×, 10×) and TG1 (6×).

FIG. 5:

Epidermal SCF causes hyperpigmentation of murine skin. (a) Newborn mouseexpressing membrane/soluble SCF (transgene one, left) shows obvioushyperpigmentation compared to non-transgenic littermate (right). (b)Transgene two positive mouse overexpressing membrane-bound epidermal SCFshows a similar phenotype with generalized hyperpigmentation which ismost discernible in the ventral and hairless areas, and which ismaintained in adult life. Three week old transgenic (left) andnon-transgenic littermate (right)

FIG. 6:

Intraepidermal melanocytes are increased in transgenic mice. (a) Tailskin section from 21 day old mouse expressing epidermal membrane-boundSCF (transgene two) shows mild epidermal hyperplasia and a markedlyincreased number of melanocytes, identified as cells surrounded by clearhalos, mostly at the dermal-epidermal junction. These mice also showextensive black epidermal melanin pigment (400×). (b) Note the lack ofboth basilar melanocytes and epidermal pigment in the skin of thetransgene (−) littermate control mouse (C57 black 6 (400×)). (c)Epidermal melanocytes express kit protein. Immunofluorescence stainingwith anti-kit antibody and Texas Red labeled secondary antibodydemonstrates confluent dendritic cells in the epidermal basalar layer ofmice expressing membrane-bound SCF (transgene two arrows). These cellscorrespond to the S-100 protein (+) basilar dendritic cells seen in FIG.2 a. Note two strongly kit positive solitary mast cells in the dermis(arrowheads, 400×). Light staining of dendritic melanocytes can also beseen in the epidermis of transgene one positive mice (please see FIG. 2b).

FIG. 7:

Electron microscopy confirms the presence of epidermal melanocytes inboth types of transgenic mice. (a) Electron microscopy shows numerouskeratinocytes containing phagocytized melanin granules in theinteradnexal epidermis of mice expressing membrane-bound epidermal SCF(3500×). b. Epidermal melanosomes, some marked with large arrows, arepresent in both keratinocytes and melanocytes. Pre melanosomes, markedwith the open arrows, demonstrate the presence of a melanocyte. Notekeratinocyte hemidesmosomes (small arrows) which confirm the location ofthe melanocyte within the epidermis (16, 320×).

FIG. 8:

Allergic ear swelling is significantly increased in SCF transgenicanimals, and is reduced by blocking the SCF receptor with the ACK2monoclonal antibody. All transgenic mice show increased ear swelling inresponse to allergic contactants compared to non-transgenic animals(p≦0.0001), showing that SCF contributes to dermatitis. Similar resultsare seen with irritant contactants (data not shown). The ear swelling isspecifically decreased by the ACK2 monoclonal antibody which blocks theSCF receptor (p≦0.05) confirming that epidermal SCF plays an active rolein cutaneous inflammation.

FIG. 9:

(a) An unpublished immunoperoxidase study of inflamed human skin with ananti-human SCF monoclonal anti-body shows soluble epidermal SCF inspongiotic (eczematous) dermatitis, here demonstrated in an epidermalspongiotic vesicle. Human spongiotic dermatitis may be associated withhyperpigmentation. Soluble epidermal SCF is not detected in normal skinwith this technique (7), suggesting that epidermal SCF is released incutaneous inflammatory states. (b) Irritant dermatitis induced intransgenic mice by Croton oil is characterized histologically byspongiotic (eczematous) dermatitis. The murine dermatitis is shown in ahematoxylin and eosin stained slide because no satisfactory antibodyagainst murine SCF is available to allow demonstration of soluble SCF byimmunoperoxidases. However, these results combined with the ACK2blocking studies shown above implicate epidermal SCF in this phenomenon.

FIG. 10:

Ear swelling response to croton oil in the 4202 (1×) strain of epidermalSCF transgenic mice. Significance levels by Mann-Whitney U Test.

FIG. 11:

Ear swelling response of the 4197 (1×) strain of transgenic mice.Significance levels use Mann-Whitney U test. Results are even moresignificant if analyzed by ANOVA with a mixed effects model (p<0.001).

FIG. 12:

Irritant dermatitis caused by croton oil is decreased by blocking KITsignaling with anti-KIT monoclonal antibody, as evidenced by decreasedear welling in 4197 (1×) SCF transgenic mice. P<0.02 by ANOVA with mixedeffects models. Please note that the ear swelling of 0.02 mm at 48 hoursin the KIT inhibited group is statistically identical to that of thenon-transgenic mice (not treated with antibody, see FIG. 10).

FIG. 13:

Effect of ACK2 on the 24-hour passive cutaneous anaphylaxis (PCA) inmice. Each amount of dye represents the mean±S.E. of 4 experiments.Treatment with saline measured O.D. of extracted dye at 0.0075±0.0015.Treatment with Anti-DNP IgE measured O.D. of extracted dye at0.0587±0.0096°. Treatment with Control IgG+Anti-DNP IgE measured O.D. ofextracted dye at 0.0571±0.0064°. Treatment with ACK2+Anti-DNP IgEmeasured O.D. of extracted dye at 0.0223±0.0049**.

-   -   *p<0.01 vs. Saline group.    -   **p<0.01 vs. anti-DNP IgE group.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method of preventing or treating in a subjectcontact dermatitis which comprises administering to the subject anamount of a compound capable of inhibiting the stem cell factorsignaling pathway effective to prevent or treat contact dermatitis so asto thereby prevent or treat contact dermatitis in the subject.

This invention provides a method of preventing or treating in a subjecthyperpigmentation which comprises administering to the subject an amountof a compound capable of inhibiting the stem cell factor signalingpathway effective to prevent or treat hyperpigmentation so as to therebyprevent or treat hyperpigmentation in the subject.

This invention also provides a method of preventing or treatingspongiotic dermatitis which comprises administering to the subject anamount of a compound capable of inhibiting the stem cell factorsignaling pathway effective to prevent or treat spongiotic dermatitis soas to thereby prevent or treat spongiotic dermatitis in the subject. Asused herein, “spongiotic dermatitis” includes but is not limited tocontact dermatitis.

This invention provides a method of preventing or treating in a subjectasthma which comprises administering to the subject an amount of acompound capable of inhibiting the stem cell factor signaling pathwayeffective to prevent or treat asthma so as to thereby prevent or treatasthma in the subject.

This invention provides a method of preventing or treating in a subjectcutaneous inflammation which comprises administering to the subject anamount of a compound capable of inhibiting the stem cell factorsignaling pathway effective to prevent or treat cutaneous inflammationso as to thereby prevent or treat cutaneous inflammation in the subject.

This invention provides a method of preventing or treating in a subjectanaphylaxis and bronchospasm which comprises administering to thesubject an amount of a compound capable of inhibiting the stem cellfactor signaling pathway effective to prevent or treat anaphylaxis andbronchospasm so as to thereby prevent or treat anaphylaxis andbronchospasm in the subject.

This invention provides a method of preventing or treating in a subjectmastocytosis which comprises administering to the subject an amount of acompound capable of inhibiting the stem cell factor signaling pathwayeffective to prevent or treat mastocytosis so as to thereby prevent ortreat mastocytosis in the subject.

This invention provides a method of preventing or treating in a subjecturticaria which comprises administering to the subject an amount of acompound capable of inhibiting the stem cell factor signaling pathwayeffective to prevent or treat urticaria so as to thereby prevent ortreat urticaria in the subject.

This invention provides a method of preventing or treating in a subjecthypersensitivity reactions which comprises administering to the subjectan amount of a compound capable of inhibiting the stem cell factorsignaling pathway effective to prevent or treat hypersensitivityreactions so as to thereby prevent or treat hypersensitivity reactionsin the subject.

This invention provides a method of preventing or treating in a subjectairway inflammation which comprises administering to the subject anamount of a compound capable of inhibiting the stem cell factorsignaling pathway effective to prevent or treat airway inflammation soas to thereby prevent or treat airway inflammation in the subject. Inone embodiment, the airway inflammation is rhinitis. In anotherembodiment, the airway inflammation is sinusitis.

This invention provides a method of preventing or treating in a subjectinterstitial cystitis which comprises administering to the subject anamount of a compound capable of inhibiting the stem cell factorsignaling pathway effective to prevent or treat interstitial cystitis soas to thereby prevent or treat interstitial cystitis in the subject. Assued herein, “interstitial cystitis” includes bladder inflammation.

This invention provides a method of preventing or treating in a subjecta tumor which expresses activated kit which comprises administering tothe subject an amount of a compound capable of inhibiting the stem cellfactor signaling pathway effective to prevent or treat a tumor whichexpresses activated kit so as to thereby prevent or treat a tumor whichexpresses activated kit in the subject.

In one embodiment, the tumor is a mast cell tumor. In anotherembodiment, the tumor is a gastrointestinal stromal tumor. In anotherembodiment, the tumor is a germ cell tumor.

In one embodiment, the above method comprises inhibiting the kinaseenzymatic reaction of kit protein.

In one embodiment, the above method comprises inhibiting chymase,elastase or other SCF cleaving enzymes.

In one embodiment, the above method comprises inhibiting ligand bindingwith an antibody, peptide, or nonpeptide chemical.

In one embodiment, the above method comprises inhibiting receptordimerization with an antibody, peptide, or nonpeptide chemical.

In one embodiment of the above method, downstream signaling of the kitactivation pathway is inhibited by blocking substrate association withthe kit kinase domain.

In one embodiment of the above method, downstream signaling of the kitactivation pathway is inhibited by blocking enzymatic function in thedownstream signaling pathway.

In one embodiment of the above method, downstream signaling of the kitactivation pathway is inhibited by blocking binding of molecules in thedownstream signaling pathway.

In one embodiment of the above method, the compound is an antibody orportion thereof. In one embodiment, the antibody is a monoclonalantibody. In one embodiment, the monoclonal antibody is a human,humanized or a chimeric antibody. In one embodiment, the monoclonalantibody is an anti-kit antibody. In one embodiment, the anti-kitantibody is ACK2.

This invention provides the above method, wherein the compound comprisesa Fab fragment of an anti-kit antibody.

This invention provides the above method, wherein the compound comprisesthe variable domain of an anti-kit antibody.

This invention provides the above method, wherein the compound comprisesone or more CDR portions of an anti-kit antibody.

This invention provides the above method, wherein the antibody isselected from the group consisting of IgA, IgD, IgE, IgG and IgM.

This invention provides the above method, wherein the compound comprisesa peptide, peptidomimetic, a nucleic acid, or an organic compound with amolecular weight less than 500 Daltons.

This invention provides the above method, wherein the compound is sSCF,sKIT ligand or a fragment thereof. As used herein “sSCF” can also mean“sKIT ligand.”

This invention provides the above method, wherein the compound is sKITor a fragment thereof.

This invention provides the above method, wherein the subject is amammal. The subject of the above methods includes but is not limited toa mammal. The subject may be a mammal or non-mammal. The subject may bea human, a primate, an equine subject, an opine subject, an aviansubject, a bovine subject, a porcine, a canine, a feline or a murinesubject. In another embodiment, the subject is a vertebrate. In apreferred embodiment, the mammal is a human being.

This invention provides the above method, wherein the administration isintralesional, intraperitoneal, intramuscular, subcutaneous,intravenous, liposome mediated delivery, transmucosal, intestinal,topical, nasal, oral, anal, ocular or otic, intravesicular, orparenteral delivery.

This invention provides a method of providing contraception to a subjectwhich comprises administering to the subject an amount of a compoundcapable of inhibiting the stem cell factor signaling pathway effectiveto prevent conception so as to thereby provide contraception to thesubject. In one embodiment, the subject is a male subject. In anotherembodiment, the subject is a female subject.

This invention provides the above method which comprises inhibiting thekinase enzymatic reaction of kit protein.

This invention provides the above method comprises inhibiting chymase,elastase or other SCF cleaving enzymes.

This invention provides a method of desensitizing a subject to an agentwhich comprises administering to the subject an amount of a compoundcapable of inhibiting the stem cell factor signaling pathway effectiveto desensitize the subject to the agent so as to thereby desensitize thesubject to the agent.

For example, the agent can be one which causes an allergic or otherimmune response in a subject. Examples of agents include but are notlimited to workplace chemicals or pollens.

One skilled in the art can employ various methods for determiningwhether a compound inhibits the stem cell signaling pathway. One ofthese methods comprises:

-   -   a) immobilizing kit protein on a solid matrix;    -   b) contacting the immobilized kit protein with the compound        being tested and a predetermined amount of SCF under conditions        permitting binding of kit protein and SCF in the absence of the        compound;    -   c) removing any unbound compound and any unbound SCF;    -   d) measuring the amount of SCF which is bound to the immobilized        kit protein;    -   e) comparing the amount measured in step (d) with the amount        measured in the absence of the compound, a decrease in the        amount of SCF bound to the kit protein in the presence of the        compound indicating that the compound inhibits binding of SCF to        kit protein, thereby indicating that the compound inhibits the        stem cell factor signaling pathway.

Another method includes detecting in vitro phosphorylation of kitcomprising the following steps:

-   -   1. Cells expressing KIT, either naturally occurring as in the        C2, BR, P815, or HMC1 lines, or as the result of DNA        transfection, are grown in vitro in the absence of exogenous        SCF.    -   2. The cells are treated with the compound or substance. In the        case of cells expressing wild type (non-mutated and not        constuitively activated) KIT, treatment with the substance or        compound may be followed by treatment with SCF. Controls include        cells treated with compound or substance, and not exposed to        SCF.    -   3. The cells are lysed and KIT is immunoprecipitated,        electrophoresed, blotted with anti-phosphotyrosine antibody, and        the antibody detected by chemiluminescence or radioactive        labeling and autoradiography, thereby determining the level of        KIT phosphorlyation on tyrosine.    -   4. The level of KIT phosphorlyation on tyrosine in cells not        exposed to the compound or substance is compared to KIT        phosphorlyation on tyrosine in cells exposed to the compound or        substance. A decrease in KIT phosphorlyation on tyrosine in the        treated cells indicates that the compound or substance inhibits        the SCF signaling pathway.

A cell proliferation and viability method comprises the following steps:

-   -   1. Cells expressing KIT, and depending on KIT activation for        survival, are grown in the presence of SCF if they express wild        type KIT or in the absence of SCF if they express mutated and        constuitively activated KIT.    -   2. The cells are grown in replicate tissue culture wells, in the        presence or absence of the compound or substance to be tested,        and the number of viable and non-viable cells per well is        determined daily by counting a sample of cells in a        hemocytometer. The number of viable and non-viable cells are        determined by the trypan blue exclusion method.    -   3. A decrease in cell growth, as determined by the presence of        fewer viable cells in the wells treated with the compound or        substance compared to the cells in the wells not so treated,        indicates that the compound or substance interferes with the KIT        SCF signaling pathway.    -   4. Alternatively, cell growth may be determined by measuring        cellular incorporation of labeled substances such as tritium        labeled thymidine.

The present invention provides a method of identifying a composition, acompound or a procedure which can produce a skin response in a subject,comprising: a) administering said composition or compound, or applyingsaid procedure to the transgenic mice which express endogenous epidermalstem cell factor, and b) analyzing the contacted skin for response.

In one embodiment of the method, the composition or compound can beadministered orally or by injection.

In another embodiment of the method, the composition or compound can beadministered topically by contacting the composition or compound withthe skin of the transgenic mice.

In another embodiment of the method, the procedure is not previouslyknown.

In another embodiment of the method, the procedure is identified by themethod.

In another embodiment of the method, the procedure is DNA vaccination.

In this invention, the skin response may be induced. This skin responseincludes but is not limited to inflammation, tanning, melanoma,carcinoma or hyperpigmentation.

In another embodiment of the method, the composition may be cosmetics,medications or skin care products.

In another embodiment of the method, the composition or compound is notpreviously known.

In yet another embodiment of the method, the composition or compound isidentified by the method.

In a further embodiment of the method, a mixture is produced forproducing a skin response comprising an effective amount of thecomposition or compound identified by the method and a suitable carrier.

The present invention also provides a method of identifying acomposition, a compound, or a procedure which can reduce or treat skinresponse in a subject, comprising: a) administering said composition orcompound, or applying said procedure to the transgenic mice whichexpress endogenous epidermal stem cell factor and which had been inducedto produce a skin response and b) analyzing the skin of said transgenicmice to determine the reduction of skin response, wherein the reductionof skin response indicates that the composition, compound, or procedurecan reduce skin response.

In one embodiment of the method, the composition or compound can beadministered orally or by injection.

In another embodiment of the method, the composition or compound can beadministered topically by contacting the composition or compound withthe skin of the transgenic mice.

In another embodiment of the method, the procedure is not previouslyknown.

In another embodiment of the method, the procedure is identified by themethod.

In another embodiment of the method, the procedure is DNA vaccination.

In another embodiment of the method, the composition or compound is notpreviously known.

In another embodiment of the method, the composition or compound isidentified by the method.

In another embodiment of the method, a mixture is produced for reducingskin response comprising an effective amount of the composition orcompound identified by the method and a suitable carrier.

In another embodiment of the method, the skin response is inflammation,tanning, skin carcinoma, melanoma or hyperpigmentation.

In another embodiment of the method, the hyperpigmentation is naturaloccurring hyperpigmentation or post inflammatory hyperpigmentation.

In another embodiment of the method, the inflammation is associated withhuman hyperpigmentation, or human hypopigmentation.

In another embodiment of the method, the subject is a mouse or ahuman-being.

In another embodiment of the method, the epidermal stem cell factortransgene encodes either a membrane bound epidermal stem cell factor ora membrane/soluble epidermal stem cell factor.

In another embodiment-of-the-method, the epidermal stem cell factortransgene encodes a membrane or soluble epidermal stem cell factor.

In another embodiment of the method, the epidermal stem cell factortransgene is cloned into a construct containing a human cytokeratin 14promotor.

In another embodiment of the method, the human cytokeratin 14 promotorcauses the expression of the stem cell factor transgene in murine skinof the basal layers of the interadnexal epidermis and the follicularepithelium.

In another embodiment of the method, the skin response of the transgenicmice can be induced by applying an irritant or an allergic dermatitisinducing agent to said skin.

In another embodiment of the method, the irritant is croton oil ordinitrofluorobenzene.

In another embodiment of the method, the croton oil ordinitrofluorobenzene are applied to the ear or the abdominal skin of thetransgenic mice; wherein the abdominal skin is either hairless orshaved.

In another embodiment of the method, the croton oil is used at aconcentration of 0.2 percent.

In another embodiment of the method, the dinitrofluorobenzene is used ata concentration of 0.5 percent in a 4:1 mixture of acetone and oliveoil.

In another embodiment of the method, the reduction or treatment ofhyperpigmentation is determined by electron microscopic analysis.

In another embodiment of the method, the compound is an epidermal stemcell factor inhibitor.

In yet another embodiment of the method, the stem cell factor inhibitoris a monoclonal antibody.

In a further embodiment of the method, the monoclonal antibody is ACK2.

The present invention further provides a method of identifying acomposition, a compound or a procedure which can reduce radiation damageto the skin of a subject, comprising: a) administering said compositionor compound, or applying said procedure to the transgenic mice whichexpress endogenous epidermal stem cell factor, b) subjecting the skin ofsaid transgenic mice and the skin of the control transgenic mice toradiation, and c) analyzing the effects of said composition, compound,or procedure on reducing skin radiation damages.

In one embodiment of the method, the composition or compound can beadministered orally or by injection.

In another embodiment of the method, the composition or compound can beadministered topically by contacting the composition or compound withthe skin of the transgenic mice.

In another embodiment of the method, the procedure is not previouslyknown.

In another embodiment of the method, the procedure is identified by themethod.

In another embodiment of the method, the procedure is DNA vaccination.

In another embodiment of the method, the composition or compound is notpreviously known.

In another embodiment of the method, the composition or compound isidentified by the method.

In another embodiment of the method, a mixture is produced for reducingskin radiation damages comprising an effective amount of the compositionor compound identified by the method and a suitable carrier.

In yet another embodiment of the method, the radiation is ultra-violetlight.

In a further embodiment of the method, the radiation damage is tanning,carcinogenesis, photo-aging, photo-damage or the development ofmelanoma.

The present invention also provides a pharmaceutical composition fortreating human skin diseases, comprising (a) a compound that can treatskin diseases of the trangenic mice which express endogenous epidermalstem cell factor, and (b) a suitable carrier, wherein the compoundspecifically targets the epidermal stem cell factor or its receptor.

In one embodiment of the pharmaceutical composition, the compound isACK2.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

Experimental Details

Transgene Construction:

Two murine SCF cDNAs were cloned into constructs containing the humancytokeratin 14 upstream region (27) (FIG. 1). This promoter causesexpression in the skin limited to the basal layers of the interadnexalepidermis and the follicular epithelium. The cDNAs were both full lengthclones, containing exon 6 encoded sequences. One cDNA (transgene one)was unmodified and therefore could produce a membrane-bound protein withthe exon 6 encoded protease sensitive site, from which a soluble,bioactive form of SCF could be efficiently generated (10, 11, 28). Theproduct of this transgene will be referred to as membrane/soluble SCF.The second cDNA (transgene two) had been previously modified by sitedirected mutagenesis, deleting the primary high efficiency cleavage site(between amino acids 164 and 165) and an alternate exon 7 encoded lowefficiency cleavage site (found in murine SCF between amino acids 180and 181). The SCF produced by this transgene therefore existspredominantly as a membrane-bound molecule (membrane SCF) (29). BothcDNAs have been previously shown to produce biologically active SCF (29,30).

Generation and Analysis of Transgenic Animals:

Two μg/ml transgenic DNA, in 10 mM Tris (pH 7.5), 0.1 mM EDTA wasinjected into fertilized oocytes collected from pseudopregnant mice asdescribed (31). At birth, most transgene expressing mice could beidentified by distinctive pigmentary phenotypes, as described in theResults section. Integration of transgenes was verified by polymerasechain reaction (PCR) of genomic DNA with transgene-specific primers andcopy number estimated by Southern blotting of PCR products, followed byautoradiography and densitometry. Skin specific expression of transgenemessenger RNA was confirmed by northern blotting and by reversetranscription-polymerase chain reaction with transgene specific primersusing RNA extracted from representative animals. Transgene expressionwas quantitated by RPA II Ribonuclease Protection Assay Kit (Ambion,Austin, Tex., USA) according to the manufacturer's directions. Briefly,total RNA extracted from mouse skin was hybridized with digoxigeninlabeled single stranded RNA probes for twenty three hours at 42° C.,digested with RNAse A and RNAse T1, electrophoresed through 5%polyacrylamide/7 Molar urea, protected fragments were transferred to Hy⁺membrane (Boerheringer-Mannheim, Indianapolis, Ind., USA), bandsdetected by chemiluminescences, and band density determined bydensitometry. Preliminary studies of RNA preparations from eachtransgenic line were performed to measure Beta-actin, and the amounts ofRNA for SCF mRNA determinations adjusted for comparison. RNA was alsoused with reverse transcription and the polymerase chain reaction fordirect amplimer sequencing of c-kit mRNA sequences in regions whichcould contain known activating mutations, as previously described (25).The primers used were 5′ CAAATC/GCATCCC/TCACACCCTGTTCAC and5′CCATAAGCAGTTGCCTCAAC which bind to nucleotides 1568→1593 and 1854→1835and 5′ TGTATTCACAGAGACTTGGC and 5′ AAAATCCCATAGGACCAGAC binding tonucleotides 2384→2403 and 2595→2576. These regions contain the codonswith both of the activating mutations, codon 559 and codon 814,respectively which have been described in human mastocytosis and in amurine mast cell line (5, 26).

Transgene one, containing the full-length unmodified SCF cDNA(membrane/soluble SCF), was injected into 100 F1 oocytes (C57 BL6×SLJ),which were implanted into six host mothers, resulting in fourindependent hyperpigmented mice, all of which were positive for thetransgene, and 40 other littermates which were pigmentary phenotypenegative and transgene negative by PCR.

Oocytes for transgene two (membrane SCF) were F1 (C57BL/6J female×SLJ/Jmale), and the offspring could be black, agouti, or white. Injection of40 embryos and implantation into six host mothers generated 48 pups, 21of which were positive for integration by PCR. Of the 25 founder miceidentified by PCR with the transgene specific primers, 3 were black, 13were agouti, and 9 were white. Five PCR positive mice (3 agouti and 2black) showed a clearly identifiable pigmentary phenotype. Given theinability of white mice to produce normal cutaneous pigment, it ispossible that there were also white founders that expressed thetransgene without the production of an obvious change in pigment.Backcrossing of phenotype positive, black and agouti founders to C57BL/6 mice produced uniform pigmentary changes, described in the Resultssection.

Histology, Immunohistochemistry, and Electron Microscopy:

Tissues from transgenic and littermate mice were fixed in formalin andembedded in paraffin or polyester wax, sectioned, and stained withhematoxylin and eosin, azure blue, alcian blue, or Giemsa's stainaccording to standard techniques (31–33). Immunofluorescence studieswere performed on polyester wax embedded sections or frozen sections,also using standard techniques. Antibodies included anti-S100 (rabbitanti-cow S100, pre-diluted, Dako, Carpinteria, Calif.), and the ACK2 andACK4 monoclonals (rat anti-mouse c-kit (34), at 20 μg/ml). Controlsincluded omission of the primary antibody or the use of isotype matchedmonoclonal antibodies of irrelevant specificity. Electron microscopy wasdone as previously described (35).

Inflammation Inducement and Treatment

We used Croton Oil and dinitrofluorobenzene (DNFB), respectively toreduce irritant and allergic contact dermatitis, respectively, inHK14-SCF transgenic mice and their non-transgenic liter-mates. CrotonOil was applied directly to the ears of mice and DNFB was applied to theears of mice after sensitization on shaved abdominal skin. Ear swellingwas measured with a micrometer. In the ear-swelling test, the transgenicmice were divided into two groups; one group was treated with the murinemonoclonal antibody ACK2, which blocks the interaction of SCF with itsreceptor (KIT), and the other group was treated with only saline. Inaddition, shaved abdominal skin of some mice was also treated withCroton Oil or with DNFB, and observed for inflammation andhyperpigmentation.

For statistical analysis, we used a mixed effects model, which allows usto fit repeated measurements over time and to compare different groupsover time. We also performed orthogonal contrasts to evaluate thedifference between treated and control groups at each time point.Immunoperoxidase study, using anti human SCF monoclonal antibodies, wereperformed in skin by standard method (7).

Experimental Results

Dermal mast cells accumulate in the presence of membrane/solublekeratinocyte SCF. Sections of skin from all mice producingmembrane/soluble SCF (transgene one) showed increased mast cells in thedermis (FIG. 2). In newborn transgene one positive mice, the mast cellswere superficial near the dermal-epidermal junction, close to theepidermal source of soluble SCF (FIG. 2 a). In older mice the mast cellsfilled the papillary dermis in some areas, but were also present in thereticular dermis, in a pattern identical to that of human mastocytosis(FIG. 2, b–d). Electron microscopic analysis confirmed the presence ofnumerous mast cells with characteristic granules within the dermis ofthe transgene one positive animals, and also showed that some of theheavily pigmented cells within the dermis of transgene one positive micewere melanocytes (FIG. 3). Mast cells were relatively rare and dermalmelanocytes were not detected in the body wall skin of non-transgeniclittermates and in transgene two positive animals of equivalent age.These observations were true across a wide range of copy numbers andlevels of SCF mRNA expression (FIG. 4). Since the keratin 14 promoter isproperly expressed in the skin only by keratinocytes, and since theproduction of only membrane-bound keratinocyte SCF did not spontaneouslyresult in increased dermal mast cells in transgene two positive animals,keratinocyte production of the soluble form of SCF appears to be able tocause cutaneous mastocytosis in mice.

SCF Transgenic Mice are Hyperpigmented:

Targeted expression of each of the SCF transgenes in murine skin causeda similar, distinctive pigmented phenotype. The pigment responsible forthe coat color of normal mice resides in the hair follicles and hairshafts, not in the epidermis. The transgenic mice, however, developedprominent epidermal pigmentation (FIG. 5). Transgene positive animalscould be identified by increased pigment at birth. By approximately 21days of age, the phenotypes were well established; phenotype positiveanimals showed pigmentation of most of the skin as well as increasedpigmentation of most of their skin as well as coat pigment. Extensivepigmentation was noted in a number of areas including the nose, mouth,ears, paws, and external genitalia when compared to normal littermatecontrols. There was enough individual variation in pigmentation so thatno clear correlation between the level of pigmentation and the levels oftransgenic expression could be shown. All transgenic animals showedsimilar degrees of pigmentation regardless of transgene type, copynumber, or levels of SCF mRNA expression. In addition to the epidermalpigmentation, the three transgene two positive agouti founders showedthin black transverse strips, consistent with the pigment distributionof the allophenic mice described by Beatrice Mintz (pictures not shown)(36).

Numerous Melanocytes are Maintained in the Skin of Transgenic Mice:

The increased pigmentation of the skin of the transgene positive mice ofboth types is attributable to the presence of intraepidermalmelanocytes, and to the epidermal melanin produced by those cells.Intraepidermal melanocytes can be identified in hematoxylin and eosinstained sections as cells in the basilar layers surrounded by clearhalos (FIG. 6, a & b) or in immunoperoxidase preparations by theirexpression of S-100 protein. Immunohistochemical analysis of animalsexpressing each of the transgenes showed numerous S-100(+)intraepidermal melanocytes (please also see FIG. 2 a). These melanocytescan be differentiated from Langerhans cells, which also express S-100protein, because melanocytes are in the basal layers and Langerhans arein the suprabasal layers. Melanocytes can also be differentiated fromLangerhans cells by their expression of the kit protein, the receptorfor SCF, which is not expressed by Langerhans cells. Staining oftransgenic animal skin with anti-kit antibody identified well-developeddendritic cells within the basilar layers of the epidermis andfollicular epithelium, consistent with melanocytes (FIGS. 6 c and 2 b).

Histologic examination confirmed the presence of pigment within theepidermis of both transgene one and transgene two phenotype positivemice from all sites examined, including the ears, tail, footpads, andbody wall (FIG. 6 a). In addition, transgene one positive mice showedmany pigmented cells within the dermis. Pigmentary abnormalities werenot observed in transgene negative littermates. Only slight epidermalpigment was identified in these control mice, and mostly in non-hairbearing areas like the footpad and tail. Although pigment patterns werestable throughout much of the adult life of the mice, an occasional TG1(msSCF) mouse developed patchy areas of depigmentation, mostly in theears, associated with loss of epidermal melanocytes and increasedpigment incontinence. This phenomenon was not observed in the mSCF mice.

Electron microscopy confirmed the presence of numerous melanocyteswithin the epidermis of both types of transgenic mice (FIG. 7).Pigmented keratinocytes, similar to those seen in the epidermis ofhumans, were also present in the interadnexal epidermis of thetransgenic mice. Intraepidermal melanocytes and pigmented keratinocyteswere extremely rare in control mice.

All Transgenic Mice Showed Ear Swelling which was Greater in Magnitudeand More Prolonged than the Non-Transgenic B6) Control Mice:

Blocking SCF by administration of ACK2 decreased the magnitude of earswelling in transgenic mice, as shown in the following FIG. 8.

Averaging Across Time:

There is a difference between the ACK2 treated and the control salinetreated transgenic mice, which is significant at the 0.05 level.Averaging across time, there is also a significant difference betweeneach of the two groups of transgenic mice (ACK2 treated and control) andthe non-transgenic mice. Both comparisons are statistically significantat the 0.0001 level. See FIG. 8. Observation of the abdominal wall skintreated with Croton Oil or DNFB showed hyperpigmentation and thickeningwhich was not observed in non-transgenic mice control (B6) mice thatwere treated identically. Histologically, hyperpigmentation correlatedwith dermal melanophages and increased epidermal melanin, identical tothe changes seen in human postinflammatory hyperpigmentation.

Discussion

Melanocytes are maintained in human epidermis throughout life. In normalmice DOPA reaction positive cells (melanoblasts and melanocytes) arefound in the epidermis at birth, but their number decreases frompostnatal day 4 and is severely reduced after one month of age (37). Onepossible explanation for the maintenance of epidermal melanocytes inhuman skin, and the difference between the distribution of melanocytesin adult human and murine skin, could be expression of epidermal SCF.Human epidermal keratinocytes produce SCF (7, 8, 39), but the SCF genedoes not appear to be expressed in murine epidermis (9). The resultspresented here show that SCF expression by murine epidermalkeratinocytes causes the maintenance and stimulation of epidermalmelanocytes throughout life. These data support the hypothesis that thedecrease in melanocyte numbers in the postnatal mouse epidermis is dueto a lack of local SCF expression. In combination with the fact that thesoluble SCF produced by Sl/Sld mice is insufficient to support normalmelanocyte survival and the observations that membrane-bound SCFpromotes longer lasting kit activation and increased survival of kitdependent cells in the hematopoietic system (40,41), our data suggestthat it is specifically the membrane-bound form of SCF that is crucialfor melanocyte survival and function.

It is interesting to note that none of the animals expressing either ofthe transgenes described in this paper have developed melanoma to date,a finding which supports previous observations that stimulation of thekit tyrosine kinase receptor does not appear to promote the developmentof melanocytic tumors (40). It also seems likely that the animalsdescribed herein, or animals derived from them, will be useful in thestudy of cutaneous mastocytosis and epidermal melanocyte biology.

The fact that SCF transgenic mice have greater responses to allergic andirritant contactants shows that epidermal SCF can actively contribute toeczematous dermatitis. This interpretation is confirmed by ourdemonstration that the inflammation can be diminished by blocking theSCF receptor with the ACK2 monoclonal antibody. Since human post natalepidermal keratinocytes express SCF, unlike post natal murine epidermalkeratinocytes, and alterations of human epidermal SCF are found inspongiotic dermatitis (a form of eczema), these observations alsosupport our contention that the skin of mice expressing epidermal SCF isa better model of human skin than is the skin of normal mice. Furthersupporting this claim is our previous observation of increased solubleepidermal SCF in the hyperpigmented lesions of mastocytosis. In sum,these data support our claim that animals expressing epidermal SCF aremore suitable for a wide variety of investigations than those which donot.

Additional Experiments

A mouse model of human skin was used in which mice express transgenicSCF driven by the human keratin 1 promotor. Normal mice do not expressepidermal SCF post-nataly, but humans and the transgenic mice do. Theepidermal SCF expressed by humans and by these mice can be solubilizedin inflammatory reactions, and based on the data produced, the solubleSCF contributes to the inflammatory process. The trends seen have beenrepeatedly confirmed in multiple different transgenic lines. Thefollowing data are representative of multiple experiments. A series oftests were conducted using croton oil as a skin irritant and 2,4dinitro-fluro-benezne (DNFB) as a contact allergen to induce cutaneousdelayed type hypersensitivity (DTH). The results shown in FIGS. 10–11show that SCF-KIT signaling is directly involved in inflammatoryresponses and implicate epidermal SCF in both irritant contactdermatitis and cutaneous DTH.

These first experiments demonstrate that irritant dermatitis caused bycroton oil is associated with increased ear swelling in epidermal SCFtransgenics compared to normal mice (non-transgenic litter mates) (FIG.10). Similar results were seen with DTH to DNFB (FIG. 11) To furthertest the hypothesis that SCF-KIT signaling contributes to thisinflammation, a monoclonal antibody was used which binds to murine KITand blocks its activation. As seen in FIGS. 12 and 8, this antibodyinhibited the exaggerated ear swelling response seen in the transgenicanimals.

It is important to note that the KIT blocking antibody can completelyblock the additional ear swelling attributable to SCF-KIT signaling inthe response to croton oil. This is by definition the efferent(effector) arm of the immune response. The fact that the same antibodytreatment does not completely abolish the allergic (DTH) response toDNFB shows, for the first time, that the afferent arm of the immuneresponse is affected by SCF-KIT signaling. Since the antibody completelyblocks the efferent effect, as evidenced by complete blocking of theresponse to croton oil, the portion of the increased transgenic responseto DNFB that is not blocked must be attributable to the afferent arm ofthe immune response. This is a novel concept. Thus, interfering withSCF-KIT signaling during the afferent, sensitization phase of the immuneresponse may be a powerful technique for preventing allergic and otheruntoward immune responses. For instance, blocking SCF-KIT signalingmight be used to induce tolerance or to desensitize individuals topotential environmental sensitizing agents such as workplace chemicalsor pollens.

Evidence for the relevance of these observations to human skin may besummarized as follows: a) Normal murine post-natal keratinocytes do notproduce SCF; b) it was previously discovered that human epidermalkeratinocytes do produce SCF, and that it is normally presentpredominantly in a cell-associated, membrane-bound form; c) Humanepidermal SCF is solubilized in the presence of neoplastic mast cells,presumably by chymase, implying that it may also be solubilized ininflammatory reactions; and d) soluble SCF staining patterns wereobserved that are similar to, but more subtle than, the solubilizationoccuring in lesions of cutaneous mastocytosis, in randomly selectedcases of human spongiotic dermatitis.

It is believed that past failures to detect a critical role for SCF-KITsignaling in DTH rest on the fact that the mice that have been studieddo not express epidermal SCF, and the contribution of SCF to murinecutaneous reactions, if there is any, has therefore been minimal. Sincehumans do express epidermal SCF, these novel and unexpected results arerelevant to human health.

We conclude that inhibition of the SCF-KIT signaling pathway has abeneficial effect in treating human dermatitis of the irritant and DTHtypes.

REFERENCES

-   1. Silvers, W. K. (1979) “The coat colors of mice: a model for    mammalian gene action and interaction” Springer-Verlag, Inc., New    York. pp. 4–5 and references therein;-   2. Mayer, T. C. (1970) “A comparison of pigment cell development in    albino, steel, and dominant-spotting mutant mouse embryos” Develop.    Biol., 23:297–309;-   3. Russell, E. S. (1979) “Hereditary anemias of the mouse: a review    for geneticists” Adv. Genet. 20:357–459;-   4. Yarden, Y., et al. (1987) “Human proto-oncogene c-kit: a new cell    surface receptor tyrosine kinase for an unidentified ligand” EMBO    J., 6:3341–3351;-   5. Qiu, F. H., et al. (1988) “Primary structure of c-kit:    relationship with the CSF-1/PDGF receptor kinase family—oncogenic    activation of v-kit involves deletion of extracellular domain and C    terminus” EMBO J., 7:1003–1011;-   6. Geissler, E. N., et al. (1988) “The dominant-white spotting (W)    locus of the mouse encodes the c-kit proto-oncogene” Cell,    55:185–192;-   7. Longley, B. J. Jr, et al. (1993) “Altered metabolism of mast-cell    growth factor (c-kit ligand) in cutaneous mastocytosis” N. Encl. J.    Med., 328:1302–1307;-   8. Weiss, R. R., et al. (1995) “Human dermal endothelial cells    express membrane-associated mast cell growth factor” J. Invest.    Dermatol., 104:101–106;-   9. Yoshida, H., et al. (1996) “Neural and skin cell specific    expression pattern conferred by Steel factor regulatory sequence in    transgenic mice” Developmental Dynamics, 207:222–232;-   10. Anderson, D. M., et al. (1990) “Molecular cloning of mast cell    growth factor, a hematopoietin that is active in both membrane bound    and soluble forms” Cell, 63:235–243;-   11. Zsebo, K. M., et al. (1990) “Identification, purification, and    biological characterization of hematopoietic stem cell factor from    Buffalo rat liver-conditioned medium” Cell, 63:195–201;-   12. Flanagan, J. G. and P. Leder (1990) “The kit ligand: a cell    surface molecule altered in steel mutant fibroblasts” Cell,    63:185–194;-   13. Onoue, H., et al. (1989) “Suppressive effects of Sl/Sld mouse    embryo-derived fibroblast cell lines on diffusible factor-dependent    proliferation of mast cells” Blood, 74:1557–1562;-   14. Anderson, D. M., et al. (1991) “Alternate splicing of mRNAs    encoding human mast cell growth factor and localization of the gene    to chromosome 12q22-q24” Cell Growth & Development, 2:373–378;-   15. Lu, H. S., et al. (1991) “Amino acid sequence and    post-translational modification of stem cell factor isolated from    buffalo rat liver cell-conditioned medium” J. Biol. Chem.,    266:8102–8107;-   16. Flanagan, J. G., et al. (1991) “Transmembrane form of the kit    ligand growth factor is determined by alternative splicing and is    missing Sld mutant” Cell, 64:1025–1035;-   17. Brannan, C. I., et al. (1991) “Steel-Dickie mutation encodes a    c-Kit ligand lacking transmembrane and cytoplasmic domains” Proc.    Natl. Acad. Sci. USA, 88:4671–4674;-   18. Zsebo, K. M., et al. (1990) “Stem cell factor is encoded at the    Sl locus of the mouse and is the ligand for the c-kit tyrosine    kinase receptor” Cell, 63:213–224;-   19. Huang, E. J., et al. (1992) “Differential expression and    processing of two cell associated forms of the kit-ligand: KL-1 and    KL-2” Mol. Biol. Cell, 3:349–362;-   20. Wehrle-Haller, B. and J. A. Weston (1995) “Soluble and    cell-bound forms of steel factor activity play distinct roles in    melanocyte precursor dispersal and survival on the lateral neural    crest migration pathway” Development, 121:731–742;-   21. Tsai, M., et al. (1991) “The rat c-kit ligand, stem cell factor,    induces the development of connective tissue-type and mucosal mast    cells in vivo: Analysis by anatomical distribution, histochemistry,    and protease phenotype” J. Exp. Med., 174:125–131;-   22. Harrist, T. J., et al. (1995) “Recombinant human stem cell    factor (SCF) (c-kit ligand) promotes melanocyte hyperplasia and    activation in vivo” Lab. Invest., 72:48A;-   23. Costa, J. J., et al. (1996) “Recombinant human stem cell factor    (KIT ligand) promotes human mast cell and melanocyte hyperplasia and    functional activation in vivo” J. Exp. Med., 183:2681–2686;-   24. Longley, B. J., et al. (1995) “The mast cell and mast cell    disease” J. Am. Acad. Dermatol., 32:545–561;-   25. Longley, B. J., et al. (1996) “Somatic c-KIT activating mutation    in urticaria pigmentosa and aggressive mastocytosis: establishment    of clonality in a human mast cell neoplasm” Nature Genetics,    12:312–314;-   6. Furitsu, T., et al. (1993) “Identification of mutations in the    coding sequence of the proto-oncogene c-KIT in a human mast cell    leukemia cell line causing ligand-independent activation of c-KIT    product” J. Clin. Invest., 92:1736–1744;-   27. Vassar, R., et al. (1989) “Tissue-specific and    differentiation-specific expression of a human K14 keratin gene in    transgenic mice” Proc. Natl. Acad. Sci. USA, 86:1563–1567;-   28. Williams, D. E., et al. (1990) “Identification of a ligand for    the c-kit proto-oncogene” Cell, 1990;63:167–174;-   29. Majumdar, M. K., et al. (1994) “Identification and mutation of    primary and secondary proteolytic cleavage sites in murine stem cell    factor cDNA yields biologically active, cell-associated protein” J.    Biol. Chem., 269:1237–1242;-   30. Yasunaga, M., et al. (1995)“Cell cycle control of c-kit-1    IL-7R1B precursor cells by two distinct signals derived from IL-7    receptor and c-kit in a fully defined medium” J. Exp. Med.,    182:315–323;-   31. Kunisada, T., et al. (1996) “Characterization and isolation of    melanocyte progenitors from mouse embryos” Development Growth &    Differentiation, 38:87–97;-   32. Yoshida, H., et al. (1996) “Distinct stages of melanocyte    differentiation revealed by analysis of nonuniform pigmentation    patterns” Development, 122:1207–1214;-   33. Scott, J. E. and R. T. Mowry (1970) “Alcian blue—a consumer's    guide” J. Histochem. Cytochem., 18:842;-   34. Nishikawa, S., et al. (1991) “In utero manipulation of coat    color formation by a monoclonal anti-c-kit antibody: two distinct    waves of c-kit-dependency during melanocyte development” EMBO J.,    10:2111–2118;-   35. Okura, M., et al. (1995) “Effects of monoclonal anti-c-kit    antibody (ACK2) on melanocytes in newborn mice” J. Invest.    Dermatol., 105:322–328;-   36. Bradl, M., et al. (1991) “Clonal coat color variation due to a    transforming gene expressed in melanocytes of transgenic mice” Proc.    Natl. Acad. Sci. USA, 88:6447–6451;-   37. Grichnik, J. M., et al. (1995) “Human recombinant stem-cell    factor induces melanocytic hyperplasia in susceptible patients” J.    Am. Acad. Dermatol., 33:577–583;-   38. Hirobe, T. (1984) “Histochemical survey of the distribution of    the epidermal melanoblasts and melanocytes in the mouse during fetal    and postnatal periods” Anat. Rec., 208:589–594;-   39. Hamann, K., et al. (1995) “Expression of stem cell factor in    cutaneous mastocytosis” Br. J. Dermatol., 133:203–208;-   40. Funasaka, Y., et al. (1992) “C-kit-kinase induces a cascade of    protein tyrosine phosphorylation in normal human melanocytes in    response to mast cell growth factor and stimulates mitogen-activated    protein kinase but is down-regulated in melanomas” Mol. Biol. Cell,    3:197–209.    Second Series

Mastocytosis is a neoplastic disease caused at least in part by somaticmutations of the c-KIT proto-oncogene resulting in constitutiveactivation of its protein product, KIT, the receptor tyrosine kinase forstem cell factor. KIT stimulates mast cell proliferation and preventsapoptosis of neoplastic mast cells. Human gastrointestinal stromal tumorcells also express mutated and activated kit (Hirota et al 1998). Todevelop potential therapies for mastocytosis and gastrointestinalstromal tumor cells we used indolinones, small molecules which inhibittyrosine kinases.

The proto-oncogene c-KIT encodes KIT (Yarden et al, 1987; Qiu et al,1988), the receptor tyrosine kinase for stem cell factor (Martin et al,1990), also known as mast cell growth factor. Somatic c-KIT mutationscausing ligand-independent activation of KIT and cell transformation(Puritsu et al, 1993; Kitayama et al, 1995; Tsujimura et al, 1996;Hirota et al, 1998; Ma et al, 1999a) appear causal in certain types ofmastocytosis (Nagata et al, 1995; Longley et al, 1996, 1999; Ma et al,1999a).

Documented activating c-KIT mutations fall into two groups. One groupconsists of mutations in codon 816 of human c-KIT, or its equivalentpositions in other species, resulting in single residue substitution forAsp816 in the activation loop of the receptor kinase domain (Ma et al,1999a). The other group of activating mutations includes single residuesubstitutions and in-frame insertions or deletions in the receptorintracellular juxtamembrane region, which disrupt intramolecularinhibition of the kinase by a putative juxtamembrane α-helix (Ma et al,1999b). All sporadic adult-onset mastocytosis patients examined to date,and a subset of pediatric cases with atypical clinical presentations,have activating codon 816 mutations (Longley et al, 1999), whereasactivating juxtamembrane mutations are common in canine mastocytomas (Maet al, 1999a) and in human gastrointestinal stromal tumors (Hirota etal, 1998).

References for Second Series

Furitsu T, Tsujimura T, Tono T, Ikeda H, Kitayama H, et al:Identification of mutations in the coding sequence of the proto-oncogenec-kit in a human mast cell leukemia cell line causing ligand-independentactivation of c-kit product. J Clin Invest 92: 1736–1744, 1993.

Hirota S, Isozaki K, Moriyama Y, Hashimoto K, Nishida T, et al:Gain-of-function mutations of c-kit in human gastrointestinal stromaltumors. Science 279: 577–580, 1998.

Kitayama H, Kanakura Y, Furitsu T, Tsujimura T, Oritani K, et al:Constitutively activating mutations of c-kit receptor tyrosine kinaseconfer factor-independent growth and tumorigenicity of factor-dependenthematopoietic cell lines. Blood 85: 790–798, 1995.

Longley B J, Tyrrell L, Lu S, Ma Y, Langley K, et al: Somatic c-KITactivating mutation in urticaria pigmentosa and aggressive mastocytosis:establishment of clonality in a human mast cell neoplasm. Nature Genet12: 312–314, 1996.

Longley B J, Metcalfe D D, Tharp M, Wang X, Tyrrell L, et al: Activatingand dominant inactivating c-KIT catalytic domain mutations in distinctclinical forms of human mastocytosis. Proc Natl Acad Sci USA 96:1609–1614, 1999.

Ma Y, Longley B J, Wang X, Blount J L, Langley K, Caughey G H:Clustering of activating mutations in c-KIT's juxtamembrane codingregion in canine mast cell neoplasms. J Invest Dermatol 112: 165–170,1999a.

Ma Y, Cunningham M E, Wang X, Ghosh I, Regan L, Longley B J: Inhibitionof spontaneous receptor phosphorylation by residues in a putativeα-helix in the KIT intracellular juxtamembrane region. J Biol Chem 274:13399–13402, 1999b.

Martin F H, Suggs S V, Langley K E, Lu H S, Ting J, et al: Primarystructure and functional expression of rat and human stem cell factorDNAs. Cell 63: 203–211, 1990.

Nagata H, Worobec A S, Oh C K, Chowdhury B A, Tannenbaum S, et al:Identification of a point mutation in the catalytic domain of theprotooncogene c-kit in peripheral blood mononuclear cells of patientswho have mastocytosis with an associated hematologic disorder. Proc NatlAcad Sci USA 92: 10560–10564, 1995.

Tsujimura T, Morimoto M, Hashimoto K, Moriyama Y, Kitayama H, et al:Constitutive activation of c-kit in FMA3 murine mastocytoma cells causedby deletion of seven amino acids at the juxtamembrane domain. Blood 87:273–283, 1996.

Yarden Y, Kuang W-J, Yang-Feng T, Coussens L, Munemitsu S, et al: Humanproto-oncogene c-kit: a new cell surface receptor tyrosine kinase for anunidentified ligand. EMBO J. 6: 3341–3351, 1987.

Third Series of Experiments

Passive Anaphylaxis: SCF and KIT stimulation have a number of effects onmast cells in vitro, but it is not clear what the overall of effects ofblocking KIT would be on inflammation and physiology in vivo. Forinstance, numerous studies have shown that recombinant SCF causes mastcell activation, directly stimulates mediator release, and alters thethreshold for IgE dependent mediator release in vitro (Nakajima et al.Biochem. Biophys. Res. Commun. 1992) (Coleman et al. J. Immunol. 1993)(Columbo et al. 1992) (Bischoff et al. J. Exp. Med. 1992). Furthermore,a single injection of recombinant SCF causes mast cell activation inmice (Werschil et al. J. Exp. Med. 1992). However, daily administrationof recombinant SCF to mice results in mast cell hyperplasia which variesat different anatomic sites, but does not result in mast cell activation(Anado et al. J. Clin. Invest. 1993), and the chronic administration ofrecombinant SCF to mice has variable effects on passive anaphylaxisreactions. Chronic stimulation with recombinant SCF followed byelicitation of passive anaphylaxis results in increased mast cellactivation at some anatomic sites but not others and, surprisingly, adecrease in deaths in mice (Anado et al. J. Clin. Invest. 1993). Thislast result is counter-intuitive and indicates that exact effects ofblocking KIT activation in vivo cannot be predicted accurately based onin vitro data. The authors themselves describe the results asunexpected.

To investigate the effects of blocking KIT in vivo, the ACK2KIT-blocking-antibody and passive cutaneous anaphylaxis were used in thefollowing experiments:

The skin of Balb/c mice was sensitized by an intradermal injection ofmurine monoclonal IgE specific for dinitrophenyl hapten. A second set ofmice was sensitized with both the IgE and ACK2. 24 hours later,dinitrophenylated human serum albumin was injected into the tail veinsof the mice in a mixture of 1% Evans' Blue dye (1 mg). Mice wereobserved for anaphylaxis, and thirty minutes later were sacrificed. Thedorsal skin at the area of sensitization was removed for measurement ofthe amount of extravasated dye. The amount of dye was determinedcolorometrically after extraction of the skin in 1 ml of 1 Normal KOHovernight, at 37 degrees centigrade. 0.6 Normal phosphoric acid inacetone (15:13) was added and the mixture cleared by centrifugation. Theabsorbent intensity (OD) of the supernatant was measured at 620 nmspectrophotometrically. The amount of extravasated dye, which is ameasure of anaphylaxis, was significantly reduced in the presence ofACK2 (mean OD 0.0223) when compared to animals sensitized without ACK2(OD 0.0617) a difference which was significant with p<0.01, and theanimals treated with ACK2 showed less respiratory distress compared toanimals without ACK2.

These data demonstrate, for the first time, that blocking the SCF-KITsignaling pathway in vivo can decrease anaphylaxis and bronchospasms.Combined with the data on cutaneous inflammation, they demonstrate thatthe SCF-KIT pathway is actively involved in inflammatory reactions invivo, rather than playing merely a supportive role in the developmentand maintenance of KIT expressing cells. This critical finding showsthat acute blockage of the SCF-KIT signaling pathway can inhibitinflammation.

Additional Experiments

A KIT blocking antibody was used to determine that SCF-KIT signalingplays an important role in vivo in passive cutaneous anaphylaxis. Thissuggests that SCF potentiation of IgE receptor signaling is important invivo. (FIG. 13). These are the first in vivo data to suggest thatblocking the SCF-KIT signaling pathway would be useful therapeuticallyfor conditions like asthma and allergic rhinitis.

References for Third Series of Experiments:

-   1. Nakajima K, Hirai K, Yamaguchi M, Takaishi T, Ohta K, Morita Y,    Ito K. Biochem. Biophys. Res. Commun. 1992, 183:1076–83.-   2. Coleman J W, Holliday M R, Kimber I, Zsebo K M and Galli S J. J.    Immunol. 1993, 150: 556–62.-   3. Columbo M, Horowitz E M, Botana L M, MacGlashan D W Jr., Bochner    B S, Gillis S, Zsebo K M, Galli S J, Lichtenstein L M. J. Immunol.    1992, 149:599–608.-   4. Bischoff S C and Dahinden Calif. J. Exp. Med. 1992, 175:237–44.-   5. Wershil B K, Tsai M, Geissler E N, Zsebo K M, Galli S J. J. Exp.    Med. 1992, 175:245–55.-   6. Ando A, Martin T R, Galli S J. J. Clin. Invest. 1993, 92:1639–49.

1. A method of preventing or treating contact dermatitis in a subjectwhich comprises administering to the subject an antibody or portionthereof that binds to kit protein in an amount effective to inhibit thekinase enzymatic reaction of kit protein and to inhibit the stem cellfactor signaling pathway, thereby preventing or treating contactdermatitis in the subject.
 2. The method of claim 1, wherein the subjectis selected from the group consisting of a cow, a horse, a sheep, a pig,a dog, a cat, a rabbit and a primate.
 3. The method of claim 1, whereinthe subject is a human being.
 4. The method of claim 1, wherein theantibody is a monoclonal antibody.
 5. The method of claim 4, wherein themonoclonal antibody is a human antibody, a humanized antibody or achimeric antibody.
 6. The method of claim 4, wherein the monoclonalantibody is an anti-c-kit antibody.
 7. The method of claim 1, whereinthe antibody is designated ACK2.
 8. The method of claim 1, wherein theadministration is intralesional, intraperitoneal, intramuscular,subcutaneous, intravenous, liposome-mediated delivery, transmucosal,intestinal, topical, nasal, oral, anal, ocular or otic delivery.